Single-cell profiling identifies impaired adaptive NK cells expanded after HCMV reactivation in haploidentical HSCT
Elisa Zaghi, Michela Calvi, Simone Puccio, Gianmarco Spata, Sara Terzoli, Clelia Peano, Alessandra Roberto, Federica De Paoli, Jasper J.P. van Beek, Jacopo Mariotti, Chiara De Philippis, Barbara Sarina, Rossana Mineri, Stefania Bramanti, Armando Santoro, Vu Thuy Khanh Le-Trilling, Mirko Trilling, Emanuela Marcenaro, Luca Castagna, Clara Di Vito, Enrico Lugli, Domenico Mavilio
Elisa Zaghi, Michela Calvi, Simone Puccio, Gianmarco Spata, Sara Terzoli, Clelia Peano, Alessandra Roberto, Federica De Paoli, Jasper J.P. van Beek, Jacopo Mariotti, Chiara De Philippis, Barbara Sarina, Rossana Mineri, Stefania Bramanti, Armando Santoro, Vu Thuy Khanh Le-Trilling, Mirko Trilling, Emanuela Marcenaro, Luca Castagna, Clara Di Vito, Enrico Lugli, Domenico Mavilio
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Research Article Hematology Immunology

Single-cell profiling identifies impaired adaptive NK cells expanded after HCMV reactivation in haploidentical HSCT

Abstract

Haploidentical hematopoietic stem cell transplantation (h-HSCT) represents an efficient curative approach for patients affected by hematologic malignancies in which the reduced intensity conditioning induces a state of immunologic tolerance between donor and recipient. However, opportunistic viral infections greatly affect h-HSCT clinical outcomes. NK cells are the first lymphocytes that recover after transplant and provide a prompt defense against human cytomegalovirus (HCMV) infection/reactivation. By undertaking a longitudinal single-cell computational profiling of multiparametric flow cytometry, we show that HCMV accelerates NK cell immune reconstitution together with the expansion of CD158b1b2jpos/NKG2Aneg/NKG2Cpos/NKp30lo NK cells. The frequency of this subset correlates with HCMV viremia, further increases in recipients experiencing multiple episodes of viral reactivations, and persists for months after the infection. The transcriptional profile of FACS-sorted CD158b1b2jpos NK cells confirmed the ability of HCMV to deregulate NKG2C, NKG2A, and NKp30 gene expression, thus inducing the expansion of NK cells with adaptive traits. These NK cells are characterized by the downmodulation of several gene pathways associated with cell migration, the cell cycle, and effector-functions, as well as by a state of metabolic/cellular exhaustion. This profile reflects the functional impairments of adaptive NK cells to produce IFN-γ, a phenomenon also due to the viral-induced expression of lymphocyte-activation gene 3 (LAG-3) and programmed cell death protein 1 (PD-1) checkpoint inhibitors.

Authors

Elisa Zaghi, Michela Calvi, Simone Puccio, Gianmarco Spata, Sara Terzoli, Clelia Peano, Alessandra Roberto, Federica De Paoli, Jasper J.P. van Beek, Jacopo Mariotti, Chiara De Philippis, Barbara Sarina, Rossana Mineri, Stefania Bramanti, Armando Santoro, Vu Thuy Khanh Le-Trilling, Mirko Trilling, Emanuela Marcenaro, Luca Castagna, Clara Di Vito, Enrico Lugli, Domenico Mavilio

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Figure 7

Impairment in the effector-functions of KIRpos mL-NK cells.

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Impairment in the effector-functions of KIRpos mL-NK cells. (A) Gene set...
(A) Gene sets involved in respiratory metabolism and enriched in KIRpos NK cells from NR and retrieved from GO. (B) Summary statistical graph showing the percentage of proliferating Ki67pos/KIRpos NK cells (%, mean ± SD) in NR and R at 7–12 months after h-HSCT. (C) Summary statistical graph showing the percentage of KIRpos/NKG2Cpos/IFN-γpos NK cells in NR (n = 3) and R (n = 3) recipients at 8–12 months after h-HSCT either in the absence (-) or presence (+) of 721.221.G target cell lines. One-way ANOVA with Bonferroni correction. (D) Summary statistical graph showing the normalized value of PDC1 and LAG3 gene expression in FACS-sorted KIRpos NK cells from NR (n = 8) and R (n = 7). Paired t test. (E) Pearson correlation between PDCD1 and LAG3 gene expression on KIRpos NK cells. (F) Summary statistical graphs showing the percentage of IFN-γpos NK cells in NR (n = 8) and R (n = 8) recipients at 8–12 months after h-HSCT, cocultured with HUVEC either in the absence (-) or presence (+) of blocking antibodies (α–PD-1 and α–LAG-3). One-way ANOVA with Bonferroni correction. (G) Summary statistical graphs showing the number of HUVEC cells infected with the BAC clone of the HCMV strain TB40/E expressing EGFP remained after 48 hours of coculture with KIRpos NK cells from NR (n = 3) and R (n = 3) either in the absence (-) or presence (+) of blocking antibodies (α–PD-1 and α–LAG-3). One-way ANOVA with Bonferroni correction. *P < 0.05, **P < 0.01, ***P < 0.001.